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Friday, February 29, 2008

[Update following this morning’s conference call: 1) Importantly, statistical analysis showed that the reduced infection rate following ALN-RSV01 administration was independent of the level of pro-inflammatory cytokines, and therefore further indicates an RNAi-based mechanism of action, instead of an siRNA-triggered non-specific cytokine response; 2) The dosages were at the upper end based on previous phase I nasal administration safety studies. There were two dosages used, a 75mg group (8/88 subjects) and a 150mg group (80/88). This number was too small, and probably too close together, to determine dose-response. 3) while I labeled the virus in my summary as a “laboratory strain”, it was stressed that the virus was a relatively fresh clinical, wild-type isolate from an RSV patient and therefore not attenuated; 4) the slides from John De Vincenzo’s presentation in Singapore will be available on the company’s website this weekend.]

Concurrent with a presentation at the respiratory disease conference in Singapore, Alnylam has just released more detailed data from the phase II experimental infection study of ALN-RSV01, an unmodified siRNA for the treatment of RSV infection via RNAi. The study, termed GEMINI, was designed to demonstrate the safety and antiviral activity of intranasally administered ALN-RSV01 in adult volunteers artificially infected with a laboratory strain of RSV virus. As such, it could therefore represent statistically validated proof-of-concept for RNAi activity in humans.

According to the press release, there were no obvious adverse effects attributable to ALN-RSV01. Although this may have been expected based on the previous phase I intranasal safety study results, considering the inflammatory potential of some siRNAs (which were apparently excluded during the pre-clinical siRNA screening process) , an siRNA in the context of a viral infection could have conceivably triggered unforeseen safety issues, even worsening rather than treating the viral infection.

On the efficacy side, ALN-RSV01 statistically reduced the infection rate across a range of laboratory parameters. When given 5 times daily, 2 days before and 3 days after viral administration, the number of volunteers remaining infection free almost doubled, from 12/42 to 24/43 treated with placebo and ALN-RSV01, respectively. Measures of viral dynamics in those patients in which viral infection took hold showed a trend towards improvement with ALN-RSV01, although they did not reach statistical significance. Similarly, symptom scores were not much different between ALN-RSV01 and placebo (at least they were not worse as may have been expected for a siRNA-triggered inflammatory response!).

It therefore appears that at least in this setting efficacy was largely an all-or-none and once viral infection took hold there was little stopping it. For a drug candidate that is designed to treat, but not prevent RSV infection (note that there are very effective preventive neutralizing antibodies for RSV on the market), this may appear disappointing at first glance.

However, there are a number of factors that complicate how predictive these results are for naturally infected patients. One unknown to me is the dose used in the study and whether they were on the conservative or aggressive side, which could have made a big difference in antiviral efficacy (but also safety, of course). Another factor is that in order to achieve reliable experimental infection, the nasal epithelium was probably overwhelmed with viral loads that would not be encountered at early stages of a natural infection. Moreover, although the experimental infection should have initially been largely restricted to the nasal epithelium, the odd survivors may be able to establish reservoirs in areas of the respiratory tract not reached by the nasal administration of the drug, at which point the route of administration became limiting in the ability of ALN-RSV01 to stop RSV replication. This may be addressed with the use of aerosolized versions of ALN-RSV01, or even in conjunction with nasal administration, in future studies.

It will also be interesting to find out whether the all-or-none response was due to viral escape mutants that have changed their sequence at the siRNA target site, as had been observed in a number of pre-clinical antiviral RNAi studies before. In that case, co-administering two different siRNAs, similar to Benitec’s HIV and HCV RNAi strategies, should considerably lower the likelihood of such an event.

Today’s results are probably almost all one could have hoped for. They are the culmination of a very well-planned and executed scientific program involving challenges such as the not-so-trivial task of establishing the experimental infection model itself. But there will be little time to rest on their laurels. The eyes are now on the design of the phase II natural infection study slated to start in the first half of this year. The all-or-none response seen here strengthens pre-clinical results suggesting that early detection and treatment will be important for the success of ALN-RSV01 of such studies, and ultimately in the clinic. With increasingly rapid nucleic acid-based diagnostics coming online, hospital-acquired cases of RSV may be the low-hanging fruit. Tomorrow’s conference call may give many of the answers, possibly linked to the enigmatic post-hoc naming of the trial as “GEMINI”.

In the larger scheme of things, the results may also have implications for the treatment and prevention of other respiratory viral infections. In terms of human proof-of-concept, with RNAi and viral infections one always has to take into account the possibility that stimulation of innate immune responses rather than specific gene silencing may have caused the antiviral effect. However, based on the preclinical studies and the fact that the drug was apparently well tolerated, one would probably have to give human proof-of-concept a pass.

Saturday, February 23, 2008

As many of you will be aware, the battle for RNAi IP is heated and particularly centers on whether Alnylam may also dominate over blunt-ended double-stranded RNAs between 22 and 24 base-pairs in length. Longer dsRNAs are covered by Fire and Mello and relatively easily accessible, 21bp and shorter dsRNAs are the domain of Kreutzer-Limmer and exclusive to Alnylam as are dsRNAs up to 25bp with 3’ overhangs (Tuschl II).

Although, even in the absence of applying obviousness criteria, I doubt that if Kreutzer-Limmer ultimately failed to be applied to 22-24bp RNAs, any other patent application would be able to do so based on prior art, this still raises the question whether 22-24bp RNAs will fall into a free-for-all grey zone.

At the end of January, Alnylam issued a press release on the issuance of the Woppmann patents in the UK (UK 2417727). Although I would not, by any stretch of the imagination, consider it to be an umbrella patent of the stature of Tuschl II, it is still a quite curious patent with relatively early 2003-4 priority in that it describes siRNAs with one blunt-end and one 3’ overhang end (often just one nucleotide) to have gene silencing advantages over the classical Tuschl design features (two 3’ overhangs, best if 2 nucleotides long).

I know that the following passage from the PR has caused some confusion among some of the readers of this blog, and admittedly also myself, as without carefully reading the claims of the patent it could be misunderstood as also covering dsRNAs with two blunt-ends, instead of dsRNAs combining the two features in just one molecule: ”The claims cover siRNA molecules of any length that contain "overhang" and "blunt end" design features, including siRNAs containing chemical modifications and certain novel motifs.”

Does this mean that Alnylam is back-tracking here on the value of 3’ overhangs, i.e. Tuschl II, which could have serious repercussions for the whole RNAi Therapeutics IP space and give companies like Silence Therapeutics or RXi some room to breath? The issue therefore is whether the one 1-nucleotide overhang is really that advantageous or just serves as a fig-leaf designed to disguise the value of blunt-end siRNAs. I therefore found the following passage from the description of another recent Alnylam patent application on the targeting of the Huntingtin gene by RNAi (USPTO application no. 11/588,674) of interest:

“In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA… dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum.”

Maybe it should not come as a surprise that with intense research, the design features of RNAi triggers will evolve over time, somewhat reminiscent of what had happened with monoclonal antibodies before and this can only be a blessing for the realization of RNAi Therapeutics. However, from a business point of view, this raises the question what will be considered sufficiently novel or merely an improvement of a fundamental design. Clearly, at a time when many dsRNAs and also single-stranded oligonucleotides are found to be able to accomplish gene silencing via RNAi, the systematic analysis of particularly dsRNAs between ~19-25bp in length with various types of overhangs or no overhangs should be of value here. I’m almost sure this has been done already and is ongoing in other parts of the industry, but it certainly would be nice to see an unbiased publication on that very subject.

Thursday, February 14, 2008

Two of medicines most popular and accepted biomarkers, LDL-cholesterol for cardiovascular disease and blood-sugar levels for diabetes, have recently come under scrutiny after high-profile studies showed no benefit or even increased health risk despite the lowering of these biomarkers by drug treatment. Since some of oligonucleotide therapeutics’ most advanced early drug candidates are for metabolic diseases and aimed at lowering these two parameters for an accelerated path into the clinic, the issue deserves some further consideration.

The ENHANCE study used an imaging technique to look at changes in the thickness of plaque deposition in the carotid (large artery running through neck) in patients treated with a combination of a statin (Merck’s “Zocor”) and the dietary cholesterol uptake inhibitor ezetimibe (“Zetia” marketed by Merck and Schering-Plough) versus patients treated with the statin alone. After a year of delay in data reporting, the companies admitted that the combination, called Vytorin, did not lead to improvements in plaque thickness with even a trend towards adverse events, despite the fact that LDL-cholesterol was further decreased by the addition of ezetimibe. Combined with Pfizer’s infamous torcetrabip blunder that despite increasing the level of good cholesterol (HDL) the rate of death was actually increased, this shows that particularly with novel drug targets, biomarkers alone will not suffice and expensive outcome studies instead may be necessary to ensure clinical benefit. A similar lesson may be drawn from the results of a recent diabetes study that aimed to aggressively lower the gold standard biomarker in diabetes care, namely blood sugar levels as measured by glycylated hemoglobin.

It will therefore be interesting whether and how this will affect the view the FDA and other regulatory agencies take on drugs such as ISIS’ ApoB100 targeting antisense compound mipomersen or Alnylam’s PCSK9-targeting ALN-PCS01 siRNA. Although there is little doubt that e.g. in the case of mipomersen, similar to Zetia, there is a marked reduction in LDL-cholesterol in humans, because it is aimed at a novel target in LDL metabolism, the FDA may insist on having its therapeutic utility being proven in large outcome studies, except maybe for some patient populations with familial hypercholesterolemia.

These failed biomarker studies are only going to add to the increasingly conservative stance the FDA takes towards drug approval, at a time when drug approval rates are declining despite ballooning drug development expenses. These expenses are also driven in part by regulatory demands for large late-stage clinical registration studies. Clearly, this trend is not compatible with a drug industry from which we expect new medicines addressing unmet medical needs. This is compounded by popular calls for cheaper generic medicines which, while saving the healthcare system dollars in the short term, undermine long-term productivity and innovation. Drug development overall has become a money-losing game for most, and it is not fair pointing out the huge cash reserves of a few Big Pharma, which we know are about to diminish in the near future anyway, without taking into account the many enterprises that never see the light of profitability.

So what could be the solution? It is difficult to argue that savings should come at the detriment of patient safety. In the difficult act of juggling the demands for patient safety, ensuring innovation, drug access, and profitability for the companies, I see fostering innovation, leading to better medicines and more efficient development paths, as probably THE one solution that may satisfy all four demands.

The RNAi Therapeutics platform e.g. opens the prospect of shortening pre-clinical development times which should lead to faster drug approval times and therefore exponentially longer periods of sales exclusivities. Say conventional drug development takes 12 out of the 15 patent years, resulting in 3 years of sales exclusivity, while an RNAi Therapeutic may take on average 9 years to develop, i.e. doubling the time of sales exclusivity. One could therefore even argue that current trends actually amplify the competitive advantages of the RNAi Therapeutics platform. Nevertheless, in the end even RNAi Therapeutics will suffer when innovation fails to be protected, which is why the current temptation for prematurely approving or tolerating unlawful generics should be resisted and fundamental patents enforced. Given the importance of the US for worldwide drug development, I can only hope the candidates running for President understand all of this.

Monday, February 11, 2008

When the Regulus CEO, during Alnylam’s quarterly conference call last week, expressed his satisfaction about their ability to attract top talent to the microRNA therapeutics start-up, Peter Linsley, who it was announced today that he would become the CSO of Regulus, must have been foremost on his mind.

Peter Linsley is the perfect CSO of Regulus in many respects. His group at Merck’s bioinformatics subsidiary Rosetta Inpharmatics was the one who seemed to abruptly end dreams of a perfectly on-target RNAi Therapeutics platform when they revealed in a 2003 paper widespread off-targeting by standard siRNAs using expression profiling (Jackson et al. 2003 “Expression profiling reveals off-target gene regulation by RNAi”). After extensive follow-up work by his group and others, we now know that much of this off-targeting is related to unmodified siRNAs being able to recognize targets based on limited base-pair complementarity in a manner essentially identical to how microRNAs recognize their targets. As such, he will bring with him to Regulus the knowledge and tools needed to predict and characterize the consequences of mimicking and inhibiting microRNAs that Regulus aims to exploit therapeutically. In addition to his bioinformatics background and experience with high-throughput technologies, he has acquired extensive knowledge on the biology of microRNAs through numerous co-authored publications.

Linsley’s know-how may also benefit the parent companies, particularly Alnylam, since his work also involved applying strategies to minimize the microRNA-like behavior of siRNAs, such as by modifying them, so that their activity would be largely limited to targets with perfect complementarity (Jackson et al. 2006 “Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing.”), therefore rendering siRNA therapeutics potentially safer. He should also bring with him expertise in identifying transcript profiles following microRNA and siRNA delivery that either indicate therapeutic promise or, even more importantly, potential harm.

Linsley’s decision to leave Merck further highlights the difficulty of Big Pharma in retaining top talent after acquiring biotech companies, particularly due to a bureaucracy that appears to be so pervasive in today’s Big Pharma and is at risk of choking innovation. Rosetta Inpharmatics and Sirna Therapeutics were part of Merck’s strategy of becoming a powerhouse in RNA therapeutics by combining leading expertise in gene regulatory networks/systems biology with oligonucleotide technologies. It now seems that much of the human capital that made these companies so attractive in the first place have left, and this, together with Alnylam’s decision last fall to terminate their RNAi Therapeutics alliance with Merck, is likely to add to the criticism that Merck overpaid for and mismanaged their acquisitions and is now at risk of losing their leadership position by insisting on going it alone.

This contrasts with Roche’s decision to let Alnylam’s former European subsidiary to operate as a semi-independent “Center of Excellence”, and is echoed by the intention of Pfizer and other Big Pharma and mature biotechs to establish similar center of innovations separate from headquarters. Of course, it is Roche’s success story with Genentech that serves as an example for these attempts.

The most remarkable aspect of today’s announcement to me, however, is the fact that Linsley personifies that by embracing complexity, what has started as a worrisome discovery 5 years ago has now turned into a therapeutic opportunity- the microRNA therapeutics opportunity.

Sunday, February 3, 2008

In the latest issue of the premier scientific journal Science, Dan Peer and colleagues from Harvard Medical School report on the successful development of an RNAi delivery system that allows for targeted gene knockdown in leukocytes following systemic (intravenous) administration (Peer et al. Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target. Science 319: 627). This research may be an important milestone towards opening up many more targets for systemic RNAi Therapeutics beyond the liver, lung, and tumors.

Until now, most systemic RNAi applications in pre-clinical animal models involved the passive delivery of various nanoparticle formulations which proved particularly effective in highly vascularized organs such as the liver. Other organs, however, are more difficult to reach mainly because most RNAi delivery formulations will either have been metabolized or excreted through the kidney, before they can reach the less accessible places in the body or enter some of the more difficult-to-transfect cell types of the blood. The present paper represents one of the more promising reports on achieving efficient gene knockdown in these organs through formulations that are actively targeted to the organ of interest through a range of peptides, antibodies, aptamers, and small molecules.

In order to combine high RNAi loading capacity, stability with consequently favorable circulation times, and cell targeting, the authors started with simple ~80nm (neutral) liposomes and added to their outside stabilizing hyaluronan (similar principle to the cationic liposome-based SNALPs- stable nucleic acids lipid particle- which typically carry stabilizing PEG on their outside). In a second step, they covalently attached an antibody specific for an integrin highly expressed on leukocytes that traffic to the gut and play a central role in autoimmune inflammatory bowel diseases such as Crohn’s. In a last step, the immunoliposome particles were loaded with the siRNA cargo that had been condensed with the highly basic protamine so as to achieve 80% loading efficiency (~4000 siRNAs per particle). That the siRNA-protamine condensation step should facilitate such efficient loading into the neutral liposomes is quite notable and may solve the poor nucleic acid loading typically associated with neutral liposomes (note: neutral liposomes may be advantageous over certain cationic liposomes in reducing unwanted interactions in the body).

The nanoparticles were then tested for gene silencing in the notoriously difficult-to-transfect leukocytes. Strikingly, sequence-specific, integrin-dependent, and antibody-dependent silencing was obtained both in vitro and in mice. The silencing efficiency was quite remarkable, typically ranging between 70-85%. When an siRNA was targeted to cyclin D1, this resulted in a reduced Th-1 response while the Th-2 response was unaffected. This is thought to be beneficial in treating chronic inflammatory diseases like Crohn’s, and sure enough, Cyclin D1 suppression by RNAi in a mouse model for intestinal inflammation almost completely reversed the disease phenotype. Hence, this paper not only demonstrates systemic gene silencing in leukocytes, but also validates Cyclin D1 as a potential drug target for Crohn’s and other inflammatory diseases.

Two aims underlie the targeted delivery paradigm: one is to deliver the RNAi trigger in sufficient amounts to the tissue of interest to achieve therapeutic levels of gene silencing; the other is to reduce potentially harmful exposure of non-target tissues to the siRNA, e.g. in instances where uptake of the siRNA in macrophages or dendritic cells may increase the risk of an unwanted immune response and render dosing less predictable, or where silencing of a gene itself in a non-target tissue may have adverse consequences. So far, most reports on targeted delivery satisfied mainly aim 1 while leaving aim 2 largely unaddressed. Quite impressively, the authors demonstrate in a biodistribution experiment truly targeted delivery by showing that the siRNA-nanoparticle were quantitatively re-directed to the gut in the mouse disease model (an almost 100-fold increase) and depleted from the blood and liver which would otherwise take up a good fraction of siRNA-nanoparticle studded with control antibody.

Of course, more time will have to be spent characterizing this system before it may move into the clinic. Safety is just one aspect that needs to be looked at in more detail, but at least in terms of cytokine activation and body weight was found to be satisfactory in the present study. Although some experiments involved repeat administration, it will further be important to determine whether there is immune recognition to any of the components of the particles which may interfere with repeat dosing. This also relates to the comparative complexity of these multifunctional particles which may also mean that the total costs of manufacturing them could considerably exceed that of the siRNA effector alone.

Surely, there will be a number of diseases for which such expenses are more than justified. I personally am not a great believer in blanketing large populations more or less indiscriminately with statins or aspirin, but rather like to see more targeted therapies that show clear benefits in well-defined patient populations. Since I expect many of the future RNAi Therapeutics to fall into the latter category and ultimately represent a bigger bang for the healthcare dollar, it is therefore important that the healthcare and patent systems facilitate rather than antagonize such innovative drug development efforts.

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